1. Field of the Invention
This invention concerns a process to convert a hydroxyl group (bold in R3C—OH) in a tigliane-type compound to a hydrogen (bold in R3C—H) to obtain deoxytigliane-type compounds or structural or functional analogs thereof. The process has wide application particularly to produce specific biologically active compounds in quantity for use as pharmaceuticals. In particular the process can be used to convert phorbol to 12-deoxytiglianes (e.g., prostratin), which are therapeutic leads for the treatment of AIDS.
2. Description of the Problems and Related Art
Availability of Prostratin
In the treatment of disease, many therapies use components from natural sources or from synthetic modifications of natural products. Aspirin was found early in the bark of a willow tree. Many natural and non-natural beta-lactam antibiotics are produced from natural sources often by semi-synthesis from a common fermentation product.1 Naturally-occurring and non-natural steroids used as therapeutics have been obtained directly or through semi-synthesis from plant sources.
More recently, taxol, an effective anti-cancer agent, was originally extracted from a natural resource, e.g., the bark and needles of the Western Yew, Taxus brevifolia. The total synthesis of taxol has been reported by the groups of Holton,2,3 Wender,4 Nicolaou,5 Danishefsky,6 Kuwajima,7 and Mukaiyama.8 While taxol is thus available through total synthesis, it has proven to be significantly more practical to produce it through semi-synthesis from a more readily available and structurally related plant natural product of the baccatin family. Semi-synthesis has also led to the production of non-natural taxanes like taxotere that have proven to be more effective in some therapeutic applications than the natural product taxol. A recent compilation of natural products as sources of new drugs indicates that a significant percentage of all new drugs introduced over the last 25 years were either natural products or derived from natural products through semi-synthesis.1 Not unlike the examples cited above and references cited therein, prostratin is currently available only in low natural abundance from Euphorbia cornigera, Homolanthus nutans and other organisms.9 Other approaches are being explored, but none has yet addressed supply. Thus, there is a need in the art for a new process for production of prostratin and structural or functional analogs thereof through semi-synthesis from readily available starting materials.
AIDS and the HIV Reservoir Problem
AIDS (acquired immune deficiency syndrome) is a pandemic disease caused by HIV (human immunodeficiency virus). In a recent report, UNAIDS (the Joint United Nations Programme on HIV/AIDS) estimated that 33.2 million people were living with HIV and 2.1 million people lost their lives to AIDS in the year 2007.10 
HAART (highly active antiretroviral therapy) has been successful in decreasing HIV-I in the plasma to undetectable levels in many treated patients. However, latent virus reservoirs remain in patients even after HAART. Such latent virus reservoirs are not targeted by current drug treatments and slowly produce the active virus over time. Therefore, interruption of treatment on such patients often results in viral rebound at a later stage.
Because HAART treats only active virus, the latent virus reservoirs decrease only very slowly in patients on HAART. It is estimated that decades of treatment would be required to deplete the latent viral reservoirs. Such long treatment is undesirable due to the side effects arising from prolonged use of the required therapeutic agents, the expense associated with this chronic therapy, patient compliance concerns, and the eventual emergence of resistance to the chronically administered therapeutics by viral mutation. Therefore, agents that can controllably flush the latent virus from its reservoirs could, in principle, provide a means to eradicate the virus when used as adjuvants in combination with HAART11.
Prostratin's Activity—A Potential Solution to the HIV Reservoir Problem
Prostratin (12-deoxyphorbol-13-acetate) (FIG. 1) is a tigliane diterpene first isolated from Pimelea prostrata and reported by Cashmore et al. in 1976.12 In 1985, Miana et al. reported isolation of prostratin from Euphorbia cornigera.13 More recently, prostratin has been found in limited quantities in the Western Samoan plant Homalanthus nutans and other organisms. FIG. 2 shows a photograph of Homalanthus nutans (left) and a Samoan healer preparing an extract from the bark of the Samoan mamala tree (right). Prostratin demonstrated multiple promising activities against HIV, which are described in the following sections.14 
Prostratin Induces Activation of Latent HIV Virus.15 
In latently infected CD4+ T cells, prostratin induces HIV gene expression. NF-κB and PKC (α and θ) activation are the key events triggered by prostratin. Although other phorbol esters such as PMA (phorbol 12-myristate-13-acetate) are also shown to activate latent HIV, prostratin differs markedly from these and offers distinct therapeutic value because it does not exhibit the tumor-promoting activity of these other agents. Therefore, prostratin is a promising therapeutic lead as an adjuvant to be used in HAART.
Prostratin Protects HIV-Infected Immune Cells from Cell Death
In an in vitro study, prostratin was shown to protect T-lymphoblastoid CEM-SS and C-8166 cell lines. At a prostratin concentration of approximately 1 μM, cell viability was restored to the level of uninfected controls, and no sign of cytotoxicity was observed up to about 25 μM. The mode of action is unclear, but the Ki of prostratin for PKC is 12 nM, suggesting the involvement of PKC in the process.14a 
Prostratin Inhibits HIV Invasion into Healthy Cells by Downregulating the Expression of HIV Receptors on Cell Surfaces16 
In CEM-SS and MT-4 cell lines, CD4 receptors were significantly reduced on cell surfaces, and mRNA array assay confirmed that CD4 gene expression along with other HIV-1 receptors (CXCR4 and CCR5) were downregulated in THP-1 cells. Staurosporine, a PKC inhibitor was shown to reverse the CD4 downregulation by prostratin, implying the involvement of PKC activation in the process. In addition, prostratin stimulates the internalization and subsequent degradation of CD4 and CXCR receptors in CEM cells. PKC translocation studies on this cell line showed PKCβ and PKCδ remained in the cytosol, whereas PKCα, τ, θ, and ε were effectively translocated to the plasma membrane.
In a more recent study, DPP (12-deoxyphorbol 13-phenylacetate), another non-tumor promoting phorbol ester, was reported to be 20-30 fold more potent than prostratin in activating latent HIV-1. DPP also downregulates CD4 and CXCR4 receptors at nanomolar concentrations.17 As is true for many therapeutic agents, greater potency could lead to improved therapeutic potential. This invention also provides a process for the production of DPP and other structural or functional analogs that may have superior clinical activity.
Potential Use of Prostratin as a Protective Adjuvant in Anticancer Radiotherapy
Activators of NF-kappaB pathway can protect healthy cells from the radiation during anticancer radiotherapy. In studies with mice and rhesus monkeys, the survival rate of the animals after radiation therapy was significantly improved when the NF-kappaB activators were injected to the animals.18 Therefore, prostratin and its functional analogs, being NF-kappaB activators without the tumor-promotion effect, hold great promise as adjuvants in anticancer radiotherapy.
Prostratin's Supply—A Hurdle in Clinical Trials and Future Human Use
Prostratin has most recently been extracted from the bark and stemwood of Samoan mamala tree (Homalanthus nutans). However, the isolation process requires multiple chromatographic separations (one Sephadex column chromatography and two HPLC purifications) and the isolation yield is very low (15 mg of prostratin from 1.05 kg of fresh stemwood).14a In addition, the prostratin content varies significantly between samples (0.2 μg/g to 52.6 μg/g).19 Other natural sources have been identified but they too suffer from low prostratin content, seasonal variation in content, and difficult separation procedures. Therefore, based on existing natural product sources, it would be difficult to economically produce prostratin in large quantities needed for human clinical trials (50 mg per treatment session).20 Such large scale harvest could place significant burden on the ecosystem. In addition, at present it is not clear that farmed trees could produce prostratin at the same levels as the wild type.
A research group at UC Berkeley signed an agreement with the Samoan government to use the mamala tree to identify and isolate the genes required to biosynthesize prostratin.23 Their goal is to engineer bacteria by inserting the genes responsible for prostratin biosynthesis and produce prostratin by fermentation. However, this technology is still in its early development stage and its feasibility is yet to be demonstrated.
These limitations on supply account for the limited research that has been done on prostratin and DPP, and the paucity of improved analogs. Accordingly, a need exists to obtain quantities of synthetic materials for use in the control of diseases.
Specific Patents and Publications
Patents of interest include but are not limited to: U.S. Pat. Nos. 5,145,842; 5,599,839; 5,643,948; 5,955,501; 6,080,784; and WO 96/40614, all of which are incorporated by reference in their entirety.